Liposomes which can carry enzymes and be labeled with antigens or antibodies are described in British Patent Application No. 8,103,282 dated Feb. 3, 1981. Liposomes labeled with antigens at their external surface and containing an enzyme entrapped in their internal volume are mixed with cognate antibody, complement, enzyme substrate and a test sample to determine whether or not the liposomes permit substrate access to the entrapped enzyme. This determination is made by detecting enzymatic activity in the presence of substrate after exposure to the test sample. Such techniques are referred to as enzyme membrane immunoassay techniques or liposome immunoassays.
A key component of the liposome immunoassay (LIA) is analyte functionalized liposome, (i.e., liposome with covalently attached antigen, antibody, or other substance of interest). Analytes can be introduced into the liposomes during their formation by incorporation of specific quantities of analyte derivatives of phosphatidylethanolamine (PEA) or other phospholipids.
Previous methods of preparation of analyte-functionalized liposomes utilize PEA-diester intermediates for direct coupling with the analyte or its derivatives. Complicated products are generated and difficulties are usually encountered in purifying and characterizing the target compound.
A method of preparing phospholipid-analyte derivatives utilizing a phosphotriester intermediate would avoid some of the difficulties encountered by the diester method. However, no triester method of forming such compounds has previously been shown in the art.
Phosphotriester intermediates, protected with benzyl groups, have been previously described, c.f., J. D. Billimoria and K. O. Lewis; J. Chem. Soc., (C), 1404 (1968). Such described intermediates are used in the preparation of phospholipids but are not applicable to preparation of phospholipid-analyte derivatives, since deprotection of such benzyl ester intermediates to the target analyte derivatized phospholipid is not compatible with most analytes required for the technology of the liposome immunoassay described above. Removal of the benzyl groups by anionic fission with sodium iodide produces benzyl iodide. That by-product is not only difficult to remove but is also a highly reactive alkylating reagent capable of reacting with any nucleophilic group on the analyte.
Use of phosphotriester methodology for different applications in phospholipid chemistry has been previously disclosed by J. H. van Boom. [C. A. A. van Boeckel et. al., Tetrahedron, 37, 3751 (1981); J. J. Oltvoort et. al., Recueil Trav. Chim. Pays-Bas, 101, 87 (1982); C. A. A. van Boeckel and J. H. van Boom, Tetrahedron Letters No. 37, 3561 (1979); C. A. A. van Boeckel and J. H. van Boom, Tetrahedron Letters, 21, 3705 (1980).] These methods disclose use of phosphotriester methodology for synthesis of naturally occurring teichoic acid fragments and modified glycophospholipids for elucidation of their function in membranes and for reasearch investigation of their physiological properties.